The substances referred to as “vitamin D” encompass a group of fat-soluble prohormones, as well as metabolites and analogues thereof. The main forms in which vitamin D occurs in the body are vitamin D2 (ergocalciferol) and vitamin D3 (cholecalciferol). The latter is the endogenous form of vitamin D, which humans can form in the skin under the influence of sunlight. The former is an exogenous form of vitamin D, taken up with food. In the US, Vitamin D2 is used as the pharmaceutical vitamin D supplement.
Whilst vitamin D2 and D3 differ in the molecular structure of their side-chains, they share the same biological activity in being prohormones, metabolized in two steps to, ultimately, 1,25 dihydroxy vitamin D (calcitriol, or 1,25 dihydroxy cholecalciferol). The preceding metabolite, 25-hydroxy vitamin D or calcidiol, results from conversion in the liver, and is considered the storage form of vitamin D in the body.
Circulating vitamin D consists mainly of 25(OH)vitamin D3 and 25(OH)vitamin D2. Biologically, 25(OH)vitamin D2 is as effective as 25(OH)vitamin D3. The half-life of 25(OH)vitamin D2 in the circulation is shorter. For clinical practice the use of an 25(OH)vitamin D assay that measures both 25(OH)vitamin D3 as well as 25(OH)vitamin D2 is recommended (1).
Vitamin D has long been recognized as an important substance, the active form of which plays a role in the formation and maintenance of bone, as well as in other processes in the human or animal body. Thus, it serves to increase the concentration of calcium in the bloodstream, by promoting absorption of calcium and phosphorus from food in the intestines, and re-absorption of calcium in the kidneys; enabling normal mineralization of bone and preventing hypocalcemic tetany. It is also necessary for bone growth and bone remodeling by osteoblasts and osteoclasts.
Vitamin D deficiency results in impaired bone mineralization and leads to bone softening diseases, rickets in children and osteomalacia in adults, and possibly contributes to osteoporosis.
In recent years it has been recognized that Vitamin D plays a number of other roles in human health. It can modulate the immune function and reduce inflammation. It has also been suggested that Vitamin D may prevent colon, breast and ovarian cancer.
Thus, it is of the essence for a person's or animal's health to have an adequate level of vitamin D.
Yet, excess of vitamin D (which may occur as a result of overdosing) is toxic. Some symptoms of vitamin D toxicity are hypercalcaemia (an elevated level of calcium in the blood) caused by increased intestinal calcium absorption. Vitamin D toxicity is known to be a cause of high blood pressure. Gastrointestinal symptoms of vitamin D toxicity can include anorexia, nausea, and vomiting. These symptoms are often followed by polyuria (excessive production of urine), polydipsia (increased thirst), weakness, nervousness, pruritus (itch), and eventually renal failure.
Clearly, it is important to be able to diagnose subjects for a possible vitamin D deficiency. It is also important, particularly for subjects that are on vitamin D supplementation, to be able to test subjects for a potential excess of vitamin D. In clinical practice, the serum level of 25-hydroxy-vitamin D is considered to be the primary indicator of the vitamin D status. (2).
Almost all circulating 25(OH)-vitamin D in serum is bound by vitamin D binding protein (88%) and Albumin (12%). Vitamin D binding protein (DBP) is an abundant protein, with a concentration of 250-400 mg/L of serum. Vitamin D is bound to DBP with a relatively high affinity, close to that of antibodies (5*108M−1).
An accurate measurement of the concentration of Vitamin D in serum requires the release of bound vitamin D from the DBP.
Early methods for the determination of Vitamin D included an extraction step using organic solvents such as acetonitrile. Other approaches have relied on dissociation of Vitamin D-DBP complex using a high or low pH (WO2004/063704). Other methods rely on the competitive displacement of Vitamin D from endogenous binding proteins using ANS (U.S. Pat. No. 7,482,162). Recently methods including proteolytic digestion of DBP have been published (WO 2008/092917 A1). Armbruster has published a method for direct measurement of Vitamin D using displacement by hydroxylated aromatic carboxylic acid (WO 2003/023391). The method described by Kyriatsoulis relies on the release of Vitamin D from Vitamin-D binding protein by using a reagent with a pH from 3.8 to 4.8 and 5-30% DMSO, a liquid organic amide and optionally 0.5-5% of a short chain alcohol. Kobold presented a method for the release based on a salt with a cation having a quaternary nitrogen based ion. EP2007/140962. US 2008/0182341 mentions stabilizing agents and capture ligands for use in assays measuring analyte concentrations. These stabilizing agents are disclosed against the background of certain alkyl amino fluoro surfactants. The inventors suggest that this surfactant facilitates the measurement of free unbound analyte versus bound analyte by stabilizing the equilibrium. Fluorocarbon octanoic acid is mentioned as a potential hazardous substance.
Background references on assaying Vitamin D include Hollis B W. Measuring 25-hydroxyvitamin D in a clinical environment: challenges and needs. Am J Clin Nutr. 2008 August; 88(2):507S-510S; Holick M F. Vitamin D: extraskeletal health. Endocrinol Metab Clin North Am. 2010 June; 39(2):381-400.